PROJECT SUMMARY/ABSTRACT Current immunotherapy strategies, including immune checkpoint blockade therapy targeting CTLA-4 and/or PD1/PD-L1, have yielded promising results in certain types of cancer patients. However, the overall success rates of these strategies still vary from 15% to 35%, which suggests that there are other mechanisms and/or checkpoint signaling involved that are unresponsive to therapy mediated by malignant tumors. Thus, alternative novel strategies targeting more specific checkpoint molecules or interrupting tolerogenic pathways are urgently needed. It is now well recognized that the suppression and dysfunction of tumor-reactive T cells induced by regulatory T cells (Treg) in the tumor suppressive microenvironment present a major barrier for successful anti-tumor immunotherapy. We recently discovered a novel suppressive mechanism whereby human Treg cells induce senescence in effector T cells that then exhibit potent suppressive activity and amplify immune suppression. Therefore, a better understanding of the cellular and molecular processes that control Treg-induced senescence in effector T cells is essential for the development of effective strategies to treat human cancer. We identified significantly increased activation of the energy sensor AMPK and dys- regulation of lipid metabolism in Treg-induced senescent T cells. Furthermore, ATM-associated DNA damage response and MAPK signaling were selectively involved in T cell senescence mediated by human Treg cells. In addition, we have discovered that human Toll-like receptor 8 (TLR8) signaling reverses the suppressive function and prevents the induction of T cell senescence mediated by both naturally occurring Treg and tumor- derived Treg cells. The central hypotheses of this proposal are that: 1) Human Treg cells can selectively modulate molecular programs that rewrite T cell lipid metabolism in treated nave/effector T cells, resulting in their differentiation into senescent T cells; 2) Senescent and dysfunctional tumor-specific T cells can be rejuvenated via checkpoint blockages of ATM and MAPK signaling in responder T cells, combined with TLR8 signaling activation in Treg cells, resulting in enhanced anti-tumor immune responses. Specific Aim 1 seeks to identify the molecular mechanism(s) responsible for the induction of senescence and dysfunction in responder T cells after interaction with Treg cells. We will dissect how Treg cells molecularly rewrite effector T cell fate and lipid metabolism. Aim 2 will test the novel concept and strategy that TLR8-mediated reprogramming of glucose metabolism in Treg cells combined with checkpoint blockage of selective MAPK and/or ATM- associated DNA damage signaling in responder T cells can synergistically enhance anti-tumor immunity through reversing the senescence and dysfunction of tumor-specific T cells. A positive outcome of these studies should lead to novel strategies to reprogram Treg metabolism and control the fate and function of tumor-specific T cells for the treatment of human cancers.